CD4 T cells are essential to the development of both cell-mediated and humoral immunity. Upon infection, naive CD4 T cells with microbial peptide-major histocompatibility complex II (MHCII)-specific T cell antigen receptors (TCR) can develop into Th1 effector cells that are specialized to activate macrophages or into T follicular helper cells (Tfh/GC-Tfh cells) that help B cells. While it is well established that the transcription fator T-bet and transcriptional repressor Bcl-6 help commit CD4 T cells to become Th1 or Tfh/GC-Tfh cells, respectively, how and why these transcriptional programs are differentially induced by nave CD4 T cells activated in the same infectious setting remains unclear. The objective of this proposal is to understand how naive CD4 T cells decide to produce differentiated effector cells. A better understanding of the mechanisms involved could lead to more intelligent vaccine design. Preliminary data indicates that differentiation of naive CD4 T cells into Th1 cells and Tfh/GC-Tfh cells is influenced by the TCR of the CD4 T cell, as effector cell populations derived from single naive cells, each with a different TCR, exhibit different patterns of Th1/Tfh/GC-Tfh differentiation, while effector cell populations derived from single monoclonal cells with the same TCR exhibit similar patterns of differentiation. Often, a clonal population of effector cells derivd from a single naive CD4 T cell is composed of both Th1 and Tfh/GC-Tfh. These data raise several questions. First, how does the TCR instruct effector CD4 T cell differentiation? We hypothesize that the strength of signaling through the TCR that leads influences a naive cell to form Th1, Tfh, and GC-Tfh populations. We will test this hypothesis by generating retrogenic CD4 T cells with antigen-specific TCRs with different sensitivities for antigen, and investigating their differentiation tendencies. Using peptide:MHCII tetramers we will also investigate the parameters of the TCR-peptide:MHCII interaction that are important for productive TCR signaling, and examine their impact on effector cell differentiation. Second, how can diverse effector progeny arise from a single naive cell? We hypothesize that differential induction of and asymmetric division of factors such as T-bet, Blimp-1, and Bcl-6 may be induced by different levels of TCR stimulation, leading to daughter cells with different effector fates. We will examine this possibility using microscopy and flow cytometry. We will also investigate whether different levels of TCR signaling induce different levels of Th1/Tfh/GC-Tfh lineage specifying molecules. These experiments could lead to a much clearer understanding of how naive CD4 T cells produced specialized effector cells. This information could be critical to the design of more effective vaccines.